Influenza PB1-F2 Inhibits Avian MAVS Signaling.

Yanna Xiao,Adam Moghrabi,Ximena Fleming-Canepa,David G Tetrault,Domingo Miranzo-Navarro,Danyel Evseev,Katharine E Magor,Chase A Stevens

doi:10.3390/v12040409

Abstract

RIG-I plays an essential role in the duck innate immune response to influenza infection. RIG-I engages the critical adaptor protein mitochondrial antiviral signaling (MAVS) to activate the downstream signaling pathway. The influenza A virus non-structural protein PB1-F2 interacts with MAVS in human cells to inhibit interferon production. As duck and human MAVS share only 28% amino acid similarity, it is not known whether the influenza virus can similarly inhibit MAVS signaling in avian cells. Using confocal microscopy we show that MAVS and the constitutively active N-terminal end of duck RIG-I (2CARD) co-localize in DF-1 cells, and duck MAVS is pulled down with GST-2CARD. We establish that either GST-2CARD, or duck MAVS can initiate innate signaling in chicken cells and their co-transfection augments interferon-beta promoter activity. Demonstrating the limits of cross-species interactions, duck RIG-I 2CARD initiates MAVS signaling in chicken cells, but works poorly in human cells. The D122A mutation of human 2CARD abrogates signaling by affecting MAVS engagement, and the reciprocal A120D mutation in duck 2CARD improves signaling in human cells. We show mitochondrial localization of PB1-F2 from influenza A virus strain A/Puerto Rico/8/1934 (H1N1; PR8), and its co-localization and co-immunoprecipitation with duck MAVS. PB1-F2 inhibits interferon-beta promoter activity induced by overexpression of either duck RIG-I 2CARD, full-length duck RIG-I, or duck MAVS. Finally, we show that the effect of PB1-F2 on mitochondria abrogates TRIM25-mediated ubiquitination of RIG-I CARD in both human and avian cells, while an NS1 variant from the PR8 influenza virus strain does not.

Highlights

Retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) are essential pattern recognition receptors for various RNA viruses, including the influenza A virus [1]
In reconstitution experiments we showed duck RIG-I is capable of interacting with chicken mitochondrial antiviral signaling (MAVS), and in initiating downstream signaling in chicken cells [11]
To confirm that duck MAVS interacts with duck RIG-I-caspase activation and recruitment domain (CARD), we investigated the co-localization of these two proteins with confocal microscopy

Summary

Introduction

RIG-I like receptors (RLRs) are essential pattern recognition receptors for various RNA viruses, including the influenza A virus [1]. The CARD domains of activated RIG-I or MDA5 bind directly to the single CARD of MAVS and nucleate a helical assembly of CARD domains from adjacent MAVS proteins, in a process called “molecular imprinting” [7]. This process promotes functional prion-like aggregates of MAVS [8], which serve as an initiation platform for a phosphorylation cascade that leads to the production of type I interferon and interferon-stimulated genes (ISGs), many of which are antiviral factors [9]

Methods

Results

Conclusion